One Protein, Two Chromophores: Comparative Spectroscopic

Article pubs.acs.org/JPCB

One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein Bernd Paulus,† Boris Illarionov,‡ Daniel Nohr,† Guillaume Roellinger,† Sylwia Kacprzak,† Markus Fischer,‡ Stefan Weber,† Adelbert Bacher,‡,§ and Erik Schleicher*,† †

Institute of Physical Chemistry, Albert-Ludwigs-University Freiburg, Albertstrasse 21, 79104 Freiburg, Germany Institute for Biochemistry & Food Chemistry, University of Hamburg, Bundesstrasse 45, 20146 Hamburg, Germany § Chemistry Department, Technical University Munich, Lichtenbergstrasse 4, 85748 Garching, Germany ‡

S Supporting Information *

ABSTRACT: We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with ∼35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensor’s principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.

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INTRODUCTION 6,7-Dimethyl-8-ribityllumazine (ribolumazine) was first identified at least 45 years ago as a biosynthetic precursor of riboflavin (Rf).1 Since then, formation of ribolumazine catalyzed by lumazine synthase and its subsequent conversion into Rf by riboflavin synthase have been studied extensively as Rf is a universally encountered cofactor in nature (for a recent review, see refs 2 and 3). An understanding of this mechanism is crucial both for commercial production via fermentation4,5 and for identification of inhibitors for the purpose of drug development since the enzymes of Rf biosynthesis are not present in human or animal hosts, which makes them potential targets for anti-infective agents.6,7 In more detail, Rf synthesis follows an exceptional pathway that includes a unique dismutation reaction of two ribolumazines, thus affording Rf and the ribolumazine precursor, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate, and is therefore of significant scientific interest. The chemical properties of ribolumazine including its redox chemistry and its potential for serving as a cofactor molecule itself, however, were treated novercally as compared to the properties of Rf and its derivatives. Therefore, only a few spectroscopic studies of the fully oxidized ribolumazine and its two other redox states, the © 2014 American Chemical Society

paramagnetic semireduced (radical) state and the fully reduced state, have been published so far: Whereas the first extensive study of lumazine radicals dates back to 1970, very little follow up has been published since then. In that study, cationic radicals of a number of structural derivatives of ribolumazine were investigated using continuous-wave (cw) electron paramagnetic resonance (EPR) at room temperature.8,9 Their signals were compared according to their hyperfine splitting patterns. However, the radicals were produced under rather harsh experimental conditions far beyond physiological relevance. The first optical absorption spectra of lumazine radicals, generated by pulse radiolysis, were recorded from aqueous solution.10 Additionally, it was demonstrated that ribolumazine phosphate can be bound to apoflavodoxin, and a radical could be generated in this complex by titration with dithionite.11 Due to its optical absorption shape it was speculated to be a neutral radical. An unresolved EPR signal was mentioned but unfortunately not shown. Received: July 29, 2014 Revised: October 17, 2014 Published: October 17, 2014 13092

dx.doi.org/10.1021/jp507618f | J. Phys. Chem. B 2014, 118, 13092−13105

The Journal of Physical Chemistry B

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Figure 1. Structure of LumP from P. leiognathi (PDB-ID 3DDY).35 The Rf cofactor from the original crystal structure was replaced by the ribolumazine cofactor from the structure of LumP from P. kishitanii (PDB-ID 3A3G)16 after structural alignment. (A) Full protein in surface representation including the cofactor that is highlighted as a stick model. (B) IUPAC nomenclature of the ribolumazine (left) and the Rf (right) cofactor. (C) Closer look into the cofactor binding site. Again, the mutated amino acids and the ribolumazine cofactor are shown as stick models. Hydrogen bonds are shown as green dashed lines. (D) Same as C but with the original Rf cofactor for comparison.

A putative anionic radical form of 6,7-(2,3-dimethylbutano)N(8)-ribityllumazine 5′-monophosphate, also generated via one-electron reduction with dithionite, was reported in complex with old yellow enzyme.12 A possible role of lumazine as prehistoric flavin-like cofactor in proteins was speculated about. In 1978, the first protein that naturally harbors a ribolumazine molecule as cofactor was isolated from the luminescent marine bacterium Photobacterium phosphoreum13,14 and consequently named lumazine protein (LumP). Later, similar proteins were isolated from other marine bacteria.15−17 LumPs are believed to act as optical transponders in the bioluminescence emission of photobacteria. These luminous bacteria use bacterial luciferases, which emit blue-green light through catalytic oxidation of reduced flavin mononucleotides (FMN) with long-chain aliphatic aldehydes.18−20 Peak emission wavelengths are located around 495 nm in isolated luciferases. Luciferase-LumP complexes, on the other hand, show blueshifted fluorescence and an increased quantum yield due to interaction with LumP.20,21 Up to 2010, LumP was believed to be the only protein with a lumazine cofactor. Since then, three studies on a subgroup of the photolyase/cryptochrome family,22−24 which is involved in light-driven DNA repair,25,26 in entrainment of the circadian clock and in a number of other light-dependent events were published.27−31 In general, members of this protein family contain two cofactors, a flavin adenine dinucleotide (FAD) that is responsible for the light-activated redox chemistry and a socalled light-harvesting cofactor that is responsible for enhancement of the reaction yield. The latter cofactor can be either a folate or a flavin derivative. In this subclade, however, a novel cofactor composition was found: aside from the essential FAD cofactor, a lumazine molecule as light-harvesting chromophore and an additional [Fe−S] cluster with yet unknown function were identified.22−24

Additional reports that the lumazine molecule is of more biological relevance than commonly anticipated came from a recent study of Brucella antisera that identified lumazine synthase as the major antigen.32 A subsequent fusion protein with a Brucella membrane protein attached to the lumazine synthase moiety has been proposed as Brucella vaccine.33 Independent of these results, further studies showed that human mucosal-associated invariant T cells expressing a T-cell receptor of low diversity recognizes especially lumazine derivatives presented by the MHC-related protein, MR1.34 These results clearly demonstrated that lumazine derivatives may turn out to be more than “only” intermediates in Rf biosynthesis, and it seems therefore likely that further (perhaps even light-active) proteins containing lumazine derivatives as cofactors (or chromophores) will be identified in the near future. Consequently, a thorough characterization of the cofactor’s photophysical and photochemical properties is indispensable. In this contribution, our in-depth spectroscopic analysis using time-resolved as well as steady-state spectroscopy with either optical or EPR detection is focused on the kinetics and characterization of the semireduced and fully reduced redox states of ribolumazine (Lurad and Lured, respectively) bound to LumP from Photobacterium leiognathi. These results will be compared with ribolumazine in solution and with Rf in its radical state bound to LumP and in solution. The advantage of this concept is that a direct comparison of both cofactors is possible as both bind identically to LumP (for an overview, the structure of LumP including both cofactors and all other amino acids under investigation are depicted in Figure 1).16,35 Additionally, several point mutants of LumP, carrying both ribolumazine and Rf cofactors, were investigated. The design of these mutants was originally based on the intramolecular sequence similarity with riboflavin synthase.36,37 For example, earlier work established that the side chains of S48 and T50 are in close proximity of the lumazine chromophore bound to the N-terminal domain of yeast riboflavin synthase. In addition, all 13093



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at 279 K with an LED (Philips Lumileds LuXEON Rebel LXML PR01 0226) emitting light at 455 ± 5 nm with a spectral irradiance of 60 ± 4 μW cm−2 nm−1 for Rf samples and an LED (Roithner LaserTechnik H2A1-H410) emitting light at 410 ± 10 nm with a spectral irradiance of 73 ± 5 μW cm−2 nm−1 for lumazine samples. The temperature was regulated to ±1 K by a temperature controller (Julabo F20-HC). UV−vis absorption spectra (typically ranging from 350 to 700 nm for Rf samples and 300 to 650 nm for lumazine samples) of each sample were measured with a double-beam UV−vis spectrophotometer (Shimadzu UV2450). Absorbance was recorded for the darkadapted system and immediately after different periods of illumination. Analysis of Photoreduction Kinetics from UV−vis Absorption Spectra. The two-step photoreduction process of the enzyme-bound cofactors was analyzed using the reaction scheme

mutant proteins showed drastically altered optical properties, thus suggesting significant modulations of protein−cofactor interactions.38

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MATERIALS AND METHODS Sample Preparation. The E. coli strain used for recombinant expression of wild-type LumP of P. leiognathi has been described elsewhere.17 Recombinant E. coli cells were grown at 310 K in LB medium containing ampicillin (100 mg/ L). Cultures were incubated under shaking to an optical density of 0.4. Isopropyl-β-D-thiogalactopyranoside was added to a concentration of 0.5 mM, and incubation was continued at 295 K for 16 h. Cells were harvested by centrifugation and stored at 193 K. Protein purification was performed at 277 K. A frozen cell mass of 5 g was thawed in 25 mL of 50 mM Tris hydrochloride, pH 7.2, containing 0.5 mM DTT (buffer A). Cells were disrupted using a FrenchPress (IUL Instruments GmbH, Königswinter, Germany). Cell debris was removed by centrifugation; the supernatant was dialyzed against 10 volumes of buffer A and centrifuged again. The supernatant was passed through a column of Q Sepharose FastFlow (1 cm × 22 cm; flow rate, 1 mL/min) that had been preequilibrated with buffer A. The column was washed with 100 mL of buffer A and developed with a linear gradient of 0−0.5 M KCl in buffer A (total volume, 320 mL). LumP was eluted from 220 to 270 mL. Fractions were combined, concentrated by ultrafiltration, and dialyzed against 50 mM sodium/potassium phosphate, pH 7.0, containing 0.02% sodium azide and 0.5 mM DTT (buffer B). The solution was centrifuged for 30 min at 20 000g, and the supernatant was passed through a column of Superdex75 (2.6 cm × 60 cm, GE Healthcare, Freiburg, Germany; flow rate, 3 mL/min). The column was developed with 360 mL of buffer B. LumP was eluted from 180 to 200 mL. Fractions were combined, concentrated by ultrafiltration, and stored at 277 K. According to SDS-PAGE, the protein sample contained less than 5% impurities. Preparation, expression, and purification of LumP mutants were described elsewhere.38 Kinetic studies and ligand exchange were performed in a buffer (0.1 M K2HPO4, pH 7.0) degassed with a turbo molecular pump and stored under an argon atmosphere. EPR samples of LumP with ribolumazine cofactor were prepared in a buffer containing 0.05 M K2HPO4, 0.01 M EDTA, pH 7.0, and 30% (v/v) glycerol (final protein concentration 1.7 mM). Wild-type protein was also transferred into a deuterated buffer (0.05 M K2HPO4, 0.01 M EDTA, pH 7.0 containing 30% (v/v) d3-glycerol in D2O) via ultrafiltration at 277 K (final protein concentration 1.8 mM). Solutions were transferred into EPR quartz tubes (3 and 0.5 mm inner diameter for X-band and W-band EPR measurements, respectively) in the dark and illuminated at 273 K. Protein samples with Rf cofactor were illuminated for 1−5 min with a halogen lamp (Streppel Halolux 100 HL); those with ribolumazine cofactor were illuminated for 3 min with an LED (Roithner LaserTechnik H2A1-H410) emitting light at 410 ± 10 nm. Afterward, samples were immediately frozen in liquid nitrogen and stored therein. UV−vis Spectroscopic Studies. For optical spectroscopy, concentrated samples were diluted to a final absorbance of 0.3− 0.6 at 450 nm for Rf samples and 420 nm for lumazine samples. All samples were prepared in a cuvette (Hellma 105.250-QS) under an argon atmosphere. Following addition of 10 mM EDTA as external electron donor,39 samples were flushed with argon gas for 5 min. Subsequently, illumination was performed

ribolumazineox or

Rf

ox

k1

HooI k −1

ribolumazine rad or

Rf

rad

k2

HooI k −2

ribolumazine red or

Rf red

where k1 and k2 are apparent rate constants for the (first-order) one-electron reduction steps of the fully oxidized cofactor, Luox or Rfox, and the cofactor’s radical state, Lurad or Rfrad, respectively. In the absence of any exogenous oxidant (such as oxygen) in the buffer solutions, the corresponding reoxidation rates, k−1 and k−2, are very small compared to k1 and k2 and have therefore been neglected in the analyses. Kinetic analysis was performed using the software Glotaran (Version 1.3).40 The models consisted of sequential decays of one or two compartments, depending on the presence or lack of the radical band in the spectra. In the case of some mutants, an additional compartment was introduced accounting for nonprotein-bound cofactor. If a radical signal was observed, the radical yield was calculated by comparing its absorbance maximum to the absorbance of the oxidized form before illumination. For this procedure, published absorption coefficients ε450 = 11 300 M−1 cm−1 for Rfox, ε580 = 4800 M−1 cm−1 for Rfrad, and ε420 = 10 100 M−1 cm−1 for Luox as well as ε530 = 2200 M−1 cm−1 for Lurad (see below) were used.15,41 Time-Resolved Optical Studies. Transient absorption spectroscopy was performed at 279 K with a commercial laserflash photolysis spectrometer (Edinburgh Instruments LP920) and recorded with a digital oscilloscope (Tektronix TDS3012C). Protein samples with concentrations of 60−120 μM were placed in a synthetic quartz (Suprasil) semimicro cell (Hellma 108F-QS). The temperature was regulated to ±0.1 K by a temperature controller (Lauda Alpha RA 8). Optical excitation was carried out with an OPO system (Continuum OPO PLUS) pumped by a Nd:YAG laser (Continuum Surelite I) at a wavelength of 460 nm for Rf samples and 430 nm for ribolumazine samples, respectively, a pulse width of approximately 6 ns, and a pulse energy of 3 mJ. The repetition rate of the spectrometer was set to 0.016 Hz. To account for probe background, transients were measured alternately with and without laser excitation and used for calculation of difference absorbance spectra with Beer−Lambert’s law. X-Band Pulsed EPR Studies. Pulsed electron−nuclear double resonance (ENDOR) spectroscopy at X-band microwave frequencies was performed with a commercial EPR spectrometer (Bruker Elexsys E680) equipped with a DICEENDOR accessory including a radiofrequency amplifier 13094



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resolution X-ray structure obtained for the LumP from P. kishitanii (PDB-ID 3A3G).16 In this structure, the ribolumazine cofactor is tethered to the narrow binding site via a manifold of hydrogen bonds. Additional stabilization is provided by several water molecules. Our model comprises eight amino acid residues, V41, S48, L49, T50, I63, D64, Q65, and A66, as well as six water molecules. All constituents of the model were directly cut out of the crystallographic structure and appropriately augmented with hydrogen atoms. The Cα of V41, the amino group of S48, the carbonyl group of T50, and the side chain of D64 were replaced by hydrogens (see Table S3, Supporting Information, for coordinates). The carbonyl group of A66 and the C(4′) carbon of the ribityl side chain of ribolumazine were replaced by methyl groups. All intermolecular structure parameters as well as the structure of the ribolumazine side chain were kept at their crystallographic positions. However, all other intramolecular parameters as well as positions of all hydrogen atoms were fully optimized. To conveniently take into account all required constraints, geometry optimization was carried out using a fragment optimization option of the ORCA program package.44 Optimization was carried out for a neutral radical state at the gradient-corrected (BP86 functional45) DFT level employing def2-TZVP Gaussian-type orbital basis sets46−48 in combination with the auxiliary Def2-TZVP/J basis sets49 for the Coulomb fitting for the resolution of the identity (RI) approximation. All hyperfine coupling constants were obtained from subsequent single-point calculations employing the B3LYP hybrid functional45,50,51 in combination with the EPRII basis set.52 All calculations were performed with ORCA.

(Amplifier Research 250A250A) and a dielectric-ring ENDOR resonator (Bruker EN4118X-MD-4), which was immersed in a helium gas-flow cryostat (Oxford CF-935). The temperature was regulated to 80 ± 1 K by a temperature controller (Oxford ITC4). Magnetic-field-swept electron-spin echo-detected EPR spectra were recorded at microwave frequencies of 9.698 (RfLumP WT), 9.713 (Rf-LumP N101W), 9.750 (Rf-LumP S48C), 9.696 (Lu-LumP WT), and 9.699 GHz (Lu-LumP WT in deuterated buffer) using a (π/2)−500 ns−π microwavepulse sequence with 60 and 120 ns π/2 and π pulses, respectively. For Davies-type ENDOR spectroscopy, a microwave pulse sequence π−t−(π/2)−τ−π with 60 and 120 ns π/2 and π pulses, respectively, and a radiofrequency pulse of 13 μs duration starting 1 μs after the first microwave pulse was used. The separation times t and τ between the microwave pulses were 15 μs and 500 ns, respectively. To avoid saturation effects due to the typically long relaxation times of flavin radicals, the entire pulse pattern was repeated with a frequency of only 400 Hz. X-Band ENDOR Analysis. Simulation of spectra was carried out using the Matlab (The MathWorks, Natick, MA) package EasySpin (using “salt” simulation routine and its builtin “esfit” fitting function).42 Please note that we assumed the angles between the g and the A tensors as collinear as no unambiguous orientation of the two tensors could be assigned yet. W-Band cw-EPR Studies. EPR spectroscopy at W-band microwave frequencies was performed with the same EPR spectrometer, now equipped with a cylindrical resonator (Bruker TeraFlex), which was immersed in a helium gas-flow cryostat. The temperature was regulated to 80 ± 1 K by a temperature controller (Oxford ITC4). Continuous-wave (cw) EPR spectra were recorded at microwave frequencies of 94.010 (Lu-LumP WT) and 94.066 GHz (Lu-LumP WT in deuterated buffer). The magnetic field axis was calibrated against a Li:LiF standard.43 Combined spectral simulations of both spectra were performed with a self-written Matlab script making use of the EasySpin simulation routine “pepper”.42 Transient EPR (trEPR) Spectroscopy. Time-resolved detection of EPR following pulsed laser excitation was performed at 80 K using a laboratory-built X-band spectrometer in conjunction with a Bruker microwave bridge (ER041 MR). Protein samples (concentration ≈ 1.5 mM) were placed in a synthetic-quartz (Suprasil) sample tube (1.8 mm inner diameter) and irradiated in a dielectric-ring resonator (Bruker ER 4118X-MD5) immersed in a laboratory-built helium gas-flow cryostat. The temperature was regulated to ±1 K by a temperature controller (Lake Shore 321). A microwave-frequency counter (EIP 548) was used to monitor the microwave frequency. Identical microwave power of 2 mW was used in all experiments. Optical excitation was carried out with an OPO system (Opta BBO-355-vis/IR) pumped by a Nd:YAG laser (Spectra Physics GCR-11) at a wavelength of 460 nm, a pulse width of approximately 6 ns, and a pulse energy of 4 mJ. The repetition rate of the laser was set to 1.25 Hz. A transient recorder (Tektronix TDS520A) with a digitizing rate of 2 ns (resolution 8 bit) was used to acquire the timedependent EPR signal. To eliminate the background signals from the laser, trEPR signals were accumulated at off-resonance magnetic-field positions (background) and subtracted from those recorded on resonance. DFT Calculations. The molecular model of ribolumazine in the binding site of LumP was prepared based on the 2.00 Å

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RESULTS Optical Spectroscopy. To monitor the photoreduction behavior of ribolumazine noncovalently bound to LumP (LuLumP), UV−vis spectra were measured at 279 K from the darkadapted sample and immediately after different periods of bluelight illumination. Figure 2 shows the blue-light-induced spectral changes in the optical absorptions of Lu-LumP in the presence of 10 mM EDTA under anaerobic conditions. In general, optical properties of ribolumazine are expected to be similar to those of Rf, despite a major blue shift of the absorption spectrum due to its smaller π system as compared to that of Rf. Therefore, we expected a two-step process involving

Figure 2. Optical absorption spectra of wild-type LumP with ribolumazine cofactor before illumination (red line), after 1 min of blue-light illumination (blue line), and after 9 min of illumination (green line). 13095



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Figure 3. Photoreduction kinetics of wild-type LumP and several mutants carrying either ribolumazine (left) or Rf (right) cofactor. Shown are the time profiles at wavelengths of maximum absorbance of the oxidized (upper) and radical (lower) state.

a radical intermediate (Lurad), finally yielding the fully reduced cofactor (Lured) upon blue-light irradiation. In accordance with the literature,15 the dark spectrum (Figure 2, red spectrum) shows the typical absorption pattern of fully oxidized protein-bound lumazine, Luox, with a broad band centered at 420 nm with no pronounced fine structure but a minor shoulder extending up to approximately 500 nm. Over the time of the experiment these intensities are drastically decreased. A new broad shoulder appears in the 470−550 nm region, reaching its peak intensity after around 1.5 min of bluelight irradiation (Figure 2, blue spectrum). This is consistent with the buildup of a radical species, Lurad, which is expected to absorb light of longer wavelengths as compared to Luox. While a precise calculation of the extinction coefficients of Lurad bound to LumP is difficult due to the overlap of multiple spectral features, data allow for a rough estimation: For Luox bound to LumP WT, an extinction coefficient of ε420 = 10 100 M−1 cm−1 was reported.15 The strongest signal at 530 nm, where only Lurad absorption is observed, is detected after 1−2 min of bluelight irradiation. Its absorbance amounts to 10−15% of the absorbance missing at 420 nm. Using the results of the global analysis of kinetic data (see also below), the signal at 530 nm can be corrected for ribolumazine molecules that are already converted into their fully reduced state. Assuming a negligible contribution of Lured to the signal at 420 nm at this point of the experiment, the extinction coefficient of LumP-bound Lurad should be on the order of ε530 ≈ 2100 M−1 cm−1.

Concomitant with a decrease of intensity of the spectral pattern attributed to the radical form of the ribolumazine cofactor, a newly formed absorption band at 320 nm builds up with prolonged illumination. Therefore, this maximum can be attributed to the final product of the photoreduction process: the fully reduced lumazine cofactor, Lured (Figure 2, green spectrum). While both reduced and radical forms of Lu-LumP are fairly stable under an argon atmosphere, their absorption signals bleach readily on exposure to air, thus restoring the Luox redox state. Kinetic Studies. To elucidate the photochemical properties of protein-bound lumazine and the influence of the protein environment in a more quantitative way, we performed photoreduction studies on wild-type LumP (LumP WT) and on a number of single-point mutant proteins carrying either ribolumazine or Rf cofactors as well as on the free cofactors in aqueous solution for comparison. In Figure 3, time profiles for both cofactors at a wavelength near the absorption maximum of the oxidized form (420 and 480 nm for ribolumazine and Rf, respectively) and a wavelength near the absorption maximum of the radical form (530 and 595 nm for ribolumazine and Rf, respectively) are shown. The decay constants calculated from these data using a global analysis are listed in Table 1. While all samples are reduced over the time of the experiment, they differ considerably in their respective reaction rates. LumP WT carrying a ribolumazine cofactor and free ribolumazine in aqueous solution are both photoreduced to Lured within the time scale of the experiment, but the 13096



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to photodegradation and consequently is not analyzed in further detail. Only in the Lu-LumP WT sample, however, a significant amount of the semireduced radical form (with a maximum yield of 35%) is accumulated (see Figure 3, lower left panel). Photoreduction speed is decreased even more in the case of the mutant Lu-LumP samples. In principle, the photoreduction behavior of the mutant Lu-LumP protein samples can be divided into two groups, Lu-LumP S48C and Lu-LumP T50E on one hand and Lu-LumP T50W and Lu-LumP N101W on the other. While the time constants of the first subclade are smaller by a factor of ∼3 as compared to LumP WT (see Table 1 for details), those of the second subclade are smaller by a factor of ∼7−8, and thus, the photoreduction is significantly slowed down. The absorbance in the spectral region of the radical absorption, however, is almost negligible for all investigated mutants. Therefore, only one time constant k1 is necessary for an exact fitting of the data (with values of 0.32, 0.44, 0.16, and 0.14 min−1 for the Lu-LumP mutants S48C, T50E, T50W, and N101W, respectively). As all data can be fitted reasonably well, we believe that our kinetic scheme is valid for all mutant samples. However, the mutant proteins LuLumP T50W and Lu-LumP N101W do not reach their fully reduced state within the time scale of the experiment, and thus, slight inconsistencies at longer times cannot be ruled out. The situation is quite different when LumP protein samples harboring the Rf cofactor are investigated under otherwise identical experimental conditions (see Figure 3, right). Here,

Table 1. Blue-Light Illumination Kinetics for LumP WT and Its Mutants Carrying Different Cofactorsa Lu

Rf

sample

k1 /min−1

k2 /min−1

radical yield /% ± 3%

free cofactor WT S48C T50E T50W N101W

3.62 1.10 0.32b 0.44b 0.16b 0.14b

0.95 0.41

3 35

k1 /min−1 7.20b 8.20 7.60 8.50b 0.53b 0.48

k2 /min−1

radical yield /% ± 3%

0.54 0.72

21 25

0.10

20

a

Listed are the apparent rate constants k1 and k2 as well as the relative radical yield, if available. bSamples fitted with a monoexponential decay; the rate constant k1 actually represents the rate-limiting reaction and not necessarily the reduction to the semireduced state.

photoreduction speed is roughly three times slower for LuLumP WT than for the cofactor in solution (see Figure 3, left). The Lu-LumP WT data can be fitted using the kinetic scheme outlined in the Methods and Materials section, application of which yield the apparent rate constants k1 = 1.10 min−1 and k2 = 0.41 min−1, whereas in the case of the free cofactor, three rate constants (using values of 3.62, 0.95, and 0.0027 min−1 for k1, k2, and k3, respectively) are required to properly fit the experimental data. The third rate constant k3 can be rationalized by a higher fraction of molecules that are subject

Figure 4. Transient absorption spectra of wild-type LumP carrying either a ribolumazine cofactor (left) or a Rf cofactor (right). Experimental conditions: Temperature 279 K, laser excitation 430 nm for ribolumazine and 460 nm for Rf. (A) Wavelength spectra of Lu-Lump at different times after laser excitation. Shown is the average of 10 time points in the range of 20−40 (dark blue) and 100−120 ns (light blue). The protein’s steadystate fluorescence spectrum (red) is shown for comparison in B. (C) Selected time signals at indicated wavelengths. All signals are true to scale. (D) Wavelength spectra of Rf-LumP at 1 (dark blue), 5 (blue), and 50 μs (light blue) after laser excitation. (E) Normalized decay-associated difference spectra (DADS) calculated by global analysis of the experimental data. For details, see text. (F) Selected time signals at indicated wavelengths. All signals are true to scale. 13097



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characterized by a species-associated difference spectrum (SADS). Three components were required for an adequate description of the time-resolved data: The first two SADS have a broad maximum at around 750 nm and can thus be assigned to the absorption of excited and relaxed flavin triplet states that decay back to the ground state with lifetimes of 3.1 and 13.1 μs, respectively (rate constants of 0.32 and 0.076 μs−1) (orange and pink lines in Figure 4E). Furthermore, a minor amount of a flavin anion radical (green line in Figure 4E) is visible with a lifetime of 360 μs (with a rate constant of 2.80 ns−1). Whether this radical is generated via a singlet- or a triplet-state precursor, it cannot be assigned unambiguously as other reaction models including various sequential decays have been tested; however, these resulted in similar rate constants, probably because of the small spectral contribution by the radical species. No additional species are necessary to fit the data. Experiments and respective global analysis were repeated using the mutant LumP samples S48C, T50E, T50W, and N101W, harboring either the ribolumazine or the Rf cofactor. Except for the mutant T50W that shows virtually no signal within our nanosecond time resolution, only moderate changes both in kinetics and in compound composition could be observed in all other samples (see Figure S1 and Tables S1 and S2, Supporting Information, for details). Transient EPR Spectroscopy. To further characterize the aforementioned intermediate flavin radicals in LumP samples containing the Rf cofactor, transient EPR experiments (trEPR) have been performed at low temperatures. Short-lived paramagnetic intermediates such as triplet states or radical pairs generated in the course of (photo)chemical reactions can be favorably studied by measuring the EPR signal intensity as a function of time at a fixed value of the external magnetic field.57,58 In contrast to conventional EPR spectroscopy, trEPR is recorded without magnetic-field modulation, thus pushing the time resolution into the nanosecond range. Similar to transient optical spectroscopy, paramagnetic species are generated by a short laser flash and detected as a function of time after pulsed excitation. To obtain spectral information, the experiment is then repeated at different magnetic-field positions. Finally, a complete trEPR spectrum comprising a two-dimensional plot of the signal intensity with respect to both the magnetic field and the time axis is obtained. In Figure 5, low-temperature trEPR spectra of Rf-LumP WT and several mutants, recorded 1 μs after laser excitation, are depicted together with their spectral simulations (see Table S1, Supporting Information, for more information). Like other spectra of flavin triplets (see, e.g.,59−61), all spectra consist of a narrow emissive feature centered at about 168.5 mT (corresponding to g ≈ 4, data not shown) and a broad signal centered at about 348 mT (g ≈ 2), spanning roughly 130 mT with emissive (E) electron-spin polarization above and enhanced absorptive (A) electron-spin polarization below g ≈ 2, respectively. The overall widths of the EPR spectra, the spectral positions of extreme and inflection points, and the electron-spin polarization patterns are typical for photogenerated flavin triplet states.59 Following the analysis outlined previously,61 the spectra show a characteristic powder pattern centered around the g value of the flavin triplet state that is dependent on the two zero-field splitting parameters D and E and the populations pi with i = x, y, z of the three triplet sublevels. In general, all samples except Rf-LumP T50W show a trEPR signal that can be fully described by a flavin triplet state. Thus, no transiently

the reduction speed of the oxidized cofactor, described by the rate constant k1, is more or less the same for the free cofactor, Rf-LumP WT, and the mutants Rf-LumP S48C and Rf-LumP T50E (with k1 values of 7.2, 8.2, 7.6, and 8.5 min−1 for the free cofactor, Rf-LumP WT, S48C, and T50E, respectively). However, only Rf-LumP WT and Rf-LumP S48C show significant accumulation of a radical intermediate (with k2 values of 0.54 and 0.72 min−1 and with maximum yields of 21% and 25%, respectively), which could be observed in the case of neither the free cofactor nor in the mutants Rf-LumP T50E and Rf-LumP T50W (see Figure 3, lower right panel). Introduction of a Trp residue into (or close to) the cofactor binding pocket, on the other hand, results in a considerable decrease of reaction speed by a factor of about 16 for Rf-LumP T50W and Rf-LumP N101W. It has to be noted that Rf-LumP N101W shows formation of a radical intermediate (with a maximum yield of 20%) but is not converted into its fully reduced state within the time scale of the experiment. Transient Absorption. To yield further insight into the molecular processes upon illumination, we performed transient absorption spectroscopy experiments on all protein samples. Fast laser spectroscopy is commonly used to examine the dynamics and reaction kinetics of molecules after light excitation. With this method, both the chemical nature of intermediates and the exact rate constants can be extracted. LumP samples, carrying either ribolumazine or Rf, were excited by a nanosecond laser pulse at 430 or 460 nm, respectively, and transient absorbance changes were monitored over a wavelength range from 370 to 730 nm for up to 100 μs after pulsed excitation, with a spectral resolution of 4 nm. Figure 4 shows two resulting spectra at different times after laser excitation and selected time traces. In the case of the LuLumP WT sample, the only observable signal is a strong emissive peak (Figure 4A), which coincides rather well with the fluorescence emission spectrum of the protein (Figure 4B). It rapidly decays within a few dozen nanoseconds corresponding to a lifetime of 36 ± 3 ns, which is in line with a value of 15.1 ns that was obtained by others at slightly lower temperature.53,54 Aside from the strong emission, no other signals are detected on a 10 μs time scale (Figure 4C). The protein as well as the mutants are known to show intense fluorescence signals under steady-state conditions.38 No additional signals that hint at radical pairs originating from an electron-transfer reaction or a metastable Lurad state can be detected. This indicates that (i) only a small fraction of the ribolumazine molecules is reduced following light excitation (the corresponding signal is then expected to be obscured by the dominant fluorescence) or (ii) the lifetime of any electron-transfer intermediates is below our time scale of about 10 ns and thus is not detectable with our setup. Significantly different spectra were obtained when Rf-LumP samples were investigated. As exemplarily depicted for RfLumP WT in Figure 4D−F, the spectrum can be divided into different parts and assigned accordingly. The negative band at about 450 nm is assigned to Rfox ground-state bleaching. Positive difference bands detected at 375−415 and 500−700 nm are attributed to excited flavin triplet states.55 No obvious signals that hint to intermediates or even to radical pairs comprising a flavin and an amino acid radical that have been identified in other flavoproteins56 could be detected. For a quantitative analysis of the Rf-LumP WT signal, we performed a global analysis of our data in terms of a kinetic scheme with parallel decaying species, in which each species is 13098



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investigation of flavoproteins in recent years (for recent reviews, see, e.g., refs 58,62, and 63); therefore, we applied this technique to characterize the radical state of the Lu-LumP WT protein in more detail. EPR experiments were started at conventional X-band frequencies (data not shown); however, the low g anisotropy of the Lurad state at these frequencies required an increase of the magnetic field up to a range where W-band frequencies are used for resonance detection. The resulting spectra, both in protonated and in deuterated buffer, are depicted in Figure 6. The anisotropy of the g tensor is rather

Figure 6. W-Band cw-EPR frozen solution spectra (black) and simulation (red) of wild-type LumP with Lu cofactor in its radical form in protonated (upper) and deuterated (lower) buffer. Spectra were recorded at 80 K with 2.1 μW microwave power and 0.1 mT modulation amplitude at 10 kHz modulation frequency and scaled to a microwave frequency of 94.010 GHz.

Figure 5. TrEPR spectra of Rf-LumP WT (A) and mutants (B−E) recorded 1 μs after pulsed laser excitation at 80 K. Each point is the average of 50 acquisitions recorded with a laser-pulse repetition rate of 1.25 Hz, at a microwave frequency of 9.68 GHz, a microwave power of 2 mW, and a detection bandwidth of 100 MHz. TrEPR spectra were generated by integration of EPR time profiles over a time window of 500 ns centered at the peak amplitude at around 1 μs after pulsed laser excitation. Red curves represent spectral simulations (see Supporting Information for details). Signals are not to scale.

small and still far from being fully resolved even by W-band EPR, a common attribute of organic π radicals.64 Additionally, there is considerable inhomogeneous line broadening resulting from hyperfine couplings (hfcs) of the electron spin to nearby nuclei. The obvious difference between protonated and deuterated sample conditions clearly indicates that some of these hfcs arise from nearby exchangeable protons. All proton hfcs included in the simulations are listed in Table 2. Proton hfcs were directly taken from ENDOR measurements (see also below) and kept constant in the fitting process to minimize the number of variable parameters. To properly reproduce the measured spectra, two additional nitrogen hfcs with axial symmetry had to be included. Their values (A|| = 42.2 MHz and A⊥ = 0, and A|| = 36.6 MHz and A⊥ = 0, respectively) were evaluated by DFT calculations. During simultaneous leastsquares fitting of protonated and deuterated spectra only the three g-tensor principal values and the line width were varied. With a line width of 1.2 mT and gx = 2.0043 ± 0.0001, gy = 2.0034 ± 0.0001, and gz = 2.0020 ± 0.0001, resulting in a giso value of 2.0032 ± 0.0001, the g tensor shows low rhombic symmetry (i.e., gx ≠ gy ≠ gz). While there are, to our knowledge, no published values from lumazine radicals for comparison, these numbers are quite similar to those from flavin radicals, both in their neutral and in their anionic states.65 In the simulations, the subtle fine features of the EPR spectra with their maxima and minima are described very well; however, minute deviations of the signal intensities between experiment and simulation are still visible in Figure 6. These can be explained by (i) a small contamination of Rf molecules in the

formed radical pairs, consisting of a flavin radical and an aminoacid radical as electron donor, can be detected for all Rf-LumP samples (on the time scale of several nanoseconds to microseconds). In more detail, spectral simulations yield values for the zero-field splitting parameters and the zero-field populations that only marginally deviate from the average values, |D| ≈ 590 × 10−4 cm−1, |E| ≈ 170 × 10−4 cm−1, px ≈ 0.57, py ≈ 0.43, and pz ≈ 0 for the samples Rf-LumP WT, T50E, and N101W (Table S2, Supporting Information). The only sample with slightly more altered triplet parameters is RfLumP S48C. Here, a value of |D| ≈ 595 × 10−4 cm−1, a larger value of |E| ≈ 195 × 10−4 cm−1, and modified sublevel populations of px ≈ 0, py ≈ 0.6, and pz ≈ 0.4 are obtained. The highly polarizable sulfur atom of the cysteine residue, located very close to the pyrazine ring of Rf, is supposed to be responsible for this effect as this amino acid could alter the symmetry of the triplet state due to sulfur−aromatic interactions. If so, one would expect an altered symmetry of the triplet state, resulting in a larger E value and modified sublevel population rates. A comparable effect has been observed in the triplet state of a methionine mutant of the photoreceptor YcgF.61 Continuous-Wave EPR Spectroscopy. Pulsed and continuous-wave paramagnetic electron resonance spectroscopy is a widely used sensitive method to detect radicals and other paramagnetic molecules. It has been very useful for 13099



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Table 2. Comparison of Experimentalb and Computedc Proton Hyperfine Couplings, Extracted from ENDOR Spectroscopya Rf cofactorb

Lu cofactor position H(3)

H(5)

Lu-H(7α) or Rf-H(8α)

H(1′)

H(1′)

hfcs A1 A2 A3 Aiso A1 A2 A3 Aiso A1 A2 A3 Aiso A1 A2 A3 Aiso A1 A2 A3 Aiso

b

wild type

deuterated buffer

b

d

0.9 (−)3.4 (−)5.5 (−)2.7 13.1 (−)15.3 (−)33.5e (−)11.9 15.8 17.1 21.3 18.1 14.3e 18.6e 21.9e 18.3e 1.3d 4.0e 6.2e 3.8e

DFT calculation 0.9 −3.3 −3.6 −2.0 10.8 −16.0 −28.1 −11.0 15.1 15.6 20.9 17.2 15.5 15.9 21.9 17.8 1.3 1.8 7.6 3.6

14.3 18.6 21.9 18.3 1.3d 4.0 6.2 3.8

c

wild type

S48C

N101W

n.d. n.d. n.d. n.d. (−)9.7 (−)25.9e (−)35.8e (−)23.8 7.3 7.9 9.4 8.2 8.9 11.5 15.6 12.0 n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. (−)9.7 (−)26.2e (−)38.7e (−)24.9 7.0 8.0 9.6 8.2 10.3 10.6 14.9 11.9 n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. (−)10.0 (−)26e (−)39e (−)25 7.0 8.1 10.0 8.4 10.4 10.7 15.1 12.1 n.d. n.d. n.d. n.d.

a All values are given in MHz. The signs of hyperfine couplings have been adapted from theoretical computations or in the case of riboflavin have been taken from the literature.65 Error margin: ±0.2 MHz; ±0.5 MHz for A3 of proton H(5). bValues result from fitted ENDOR spectra depicted in Figure 4. cB3LYP/EPR-II//RI-BP86/def2-TZVP. dTaken from DFT calculations. eValues were optimized manually.

LumP samples, (ii) small phase shifts due to the long recording times of the spectra, and (iii) small signals resulting from resonator background. All possible explanations are not expected to significantly alter the extracted EPR parameters and have been ignored in further analyses. ENDOR Spectroscopy of Lu-LumP WT. ENDOR spectroscopy is derived from EPR spectroscopy and used routinely to determine the geometric and electronic structure of paramagnetic entities from hyperfine interactions between nuclear magnetic moments and the magnetic moment of the unpaired electron spin that are too small to be resolved in the cw-EPR spectrum. Via the hyperfine coupling constant, the electron-spin density at the positions of magnetic nuclei can be evaluated. In brief, ENDOR signals are usually grouped around the nuclear Larmor frequency νn, 14.7 MHz in the case of hydrogen nuclei. In the weak-coupling limit (|vn| > |A/2|), hfcs with the orientation-dependent coupling constants A give rise to symmetrical pairs of absorption lines with distances to νn of ±A/2. Characteristic low-temperature, Davies-type X-band proton ENDOR spectra of LumP protein samples harboring either a ribolumazine or a Rf cofactor in their radical state are shown in Figure 7. For improved readability, only the part between 14 and 37 MHz is depicted. In general, the spectrum of Lu-LumP WT (Figure 7A) consists of two broad sets of resonances with tensorial shape: one set with hfcs of around 0−4 MHz and one prominent broad set with hfcs of around 12−20 MHz. The former so-called matrix signal usually comprises hfcs from protons whose nuclear spins interact only weakly with the unpaired electron spin, e.g., protons from the protein backbone within the cofactor binding pocket, protons of water molecules surrounding the ribolumazine, and also weakly coupled protons attached to ribolumazine, namely, H(3), the three hydrogens attached to the methyl group C(6α), and one of the two hydrogens attached to the ribityl carbon C(1′). Therefore, the

Figure 7. X-Band pulsed (Davies) ENDOR spectra (black) of different LumP species in their radical form and simulations (red). All spectra were recorded at 80 K. For details, see text.

three hydrogens attached to the methyl group C(7α), the second of the two hydrogens attached to the ribityl carbon C(1′), and the proton H(5) (if the radical is in its protonated 13100



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MHz, however, fits very well with the DFT calculations; thus, we believe that the error bars are reasonable. ENDOR Rf-LumP and Mutants. Characteristic ENDOR spectra of a protein with a flavin cofactor in its radical form can, in general, be grouped into five spectral regions: (i) The aforementioned matrix-ENDOR signal extends from about 13 to 16 MHz and comprises hfcs from weakly coupled protons. (ii) Signals arising from the hyperfine coupling of the H(6) proton are found at around 17 MHz. (iii) Prominent features of axial shape are observed in the flanking 17−19 MHz radiofrequency range and arise from the hfcs of protons of the methyl group attached to C(8).70−74 (iv) Flanking the H(8α) signals at around 19−20 MHz, the transitions of one of the two β protons, H(1′), are found. Finally, (v) the broad, rhombic feature extending from 21 to 34 MHz can be assigned to the exchangeable proton bound to N(5).68,69 With this knowledge at hand, the spectral simulations of RfLumP WT and the two mutants, where a radical state could also be observed (Rf-LumP S48C and Rf-LumP N101W), lead to the assignment of resonances of the protons H(8α), H(6), H(5), and H(1′) (see Figure 7C−E and Table 2). Qualitatively, the wild type and the two mutants show comparable hfc values; however, small changes in their hf patterns can be detected upon closer inspection. Whereas the hfcs of the H(8α) and H(5) protons are identical within the experimental error, clear differences are observed in the principal hfc values of H(1′): Both mutant samples reveal a more axial shape as compared to the wild type, and the A2 values are decreased by almost 1 MHz. This finding can be rationalized by a slightly different angle of the H(1′) protons with respect to the Rf’s π system. As known for other organic radicals, the strength of the hfcs of β protons follows the Mc Connell equation75 that predicts a cos2(θ) relation between the strength of the hfc and the dihedral angle theta (between the C(1′)−H bond and the 2pz orbital at N(10), both projected on a plane perpendicular to the N(10)−C(1′) bond). Therefore, mutating the amino acids S48 and N101 seems to induce small but distinct conformational changes that can be rationalized in a reorientation of hydrogen bonds between the two amino acids and the ribityl side chain of the cofactors (Figure 1).

neutral state) are expected to be located within the second peak, thus rendering the assignment quite difficult. To overcome this unfortunate situation, a second Lu-LumP WT sample in deuterated buffer was investigated under otherwise identical conditions (Figure 7B). Additionally, DFT calculations using structural information from the crystal structure (PDB-ID 3A3G,10 as outlined in more detail in the Materials and Methods section) were performed to facilitate assignment of the proton hfcs. ENDOR signals confirm the marked differences between protonated and deuterated sample conditions as all proton couplings that exchange at pH 7.0 are missing in the spectrum. Even in this case, the “matrix” region contains multiple overlapping contributions from weakly coupled protons. When comparing signals from protonated and deuterated buffer conditions, however, a small absorption line stands out in the spectrum of the protonated sample at a radiofrequency of around 16.5 MHz (Figure 7B). This signal can only arise from an exchangeable proton with small hfcs (around 3−4 MHz) and, thus, is assigned to the proton H(3). Due to the fact that in the matrix region a number of resonances are overlapping, the A1 value had to be directly taken from DFT calculations. The other values were fitted and yielded values of A2 = (−)3.4 MHz and A3 = (−)5.5 MHz. The part of the spectrum at radiofrequencies higher than 18 MHz is far more informative, because only signals of a few strongly coupled nuclei are observed in this range. In the case of Lu-LumP WT in deuterated buffer, the broad signal at radiofrequencies between 21 and 27 MHz can be described by a single hyperfine tensor. In protonated buffer, however, this signal is superimposed by additional resonances. One of them has a much stronger signal intensity and peaks at 23 MHz; the other one is very broad, reaching up to radiofrequencies of 29 MHz. The assignment of proton hfcs is guided by DFT calculations as well as the certainty thataside from the nitrogen-attached protons H(3) and H(5)the three methyl group protons H(7α) are also acidic and thus exchanged under these conditions.66 Thus, the only two protons that are remaining in deuterated buffer conditions are the ones attached to C(1′). Two rhombic hfc tensors, one with principal values of A1 = 14.3 MHz, A2 = 18.6 MHz, and A3 = 21.9 MHz, yielding Aiso = 18.3 MHz, and the other one with principal hfc values of A1 = 1.3 MHz, A2 = 4.0 MHz, A3 = 6.2 MHz, yielding an Aiso = 3.8 MHz, can be assigned (Table 2). The H(7α) protons are assigned based on the following assumptions: (i) the hyperfine tensor of a freely rotating methyl group is the average of the three individual proton hyperfine tensors similar to the respective methyl hfcs of other flavocoenzymes and (ii) the methyl-group protons are freely rotating at the temperature of the experiment.67 Assuming a nearly axial tensor with principal components of A1 = 15.8 MHz, A2 = 17.1 MHz, and A3 = 21.3 MHz, yielding Aiso = 18.1 MHz, the signal could be fitted very well. The remaining broad, rhombic feature extending up to radiofrequencies of 34 MHz in the protonated ENDOR spectrum is thus assigned to the proton bound to N(5).68,69 Its contribution to the overall spectrum is easily discriminated due to its exchangeability upon deuteration (Figure 7B). This finding unambiguously identifies the Lu radical as being in the neutral, protonated state. Because the resonance shows no clear tensorial shape and only one of the principal values (A3 = (−)33.5 MHz) is detected in the spectrum, the two other principal values (A1 = (−)13.1 and A2 = (−)15.3 MHz) have to be treated with some care. The resulting value Aiso = (−)11.9

■

DISCUSSION Optical Spectroscopy. In the present study the photoreduction kinetics of LumP WT and a series of mutants in the close vicinity of the cofactor that can either be a ribolumazine or a Rf are examined. Steady-state photoreduction kinetics were recorded under anaerobic conditions. An in-depth analysis of the data reveals that Lu-LumP WT harbors Luox as a stable species in the dark, which is converted rapidly to Lured via Lurad upon blue-light illumination (Figures 2 and 3). The fully reduced LumP reverts spontaneously to its oxidized state in the presence of oxygen. To quantify differences in the photoreaction kinetics between the various LumP samples, photoreduction reactions were analyzed assuming the presence of the three cofactor redox states and the kinetic scheme outlined in the Materials and Methods section. Analyzing the extracted rate constants k1 and k2 (Table 1), a number of important conclusions can be drawn. First, the reduction speed reflected by the rate constant k1 is in general significantly enhanced when Rf is used as cofactor. The enhancement factors range from ∼7.5 when the two WT proteins are compared up to ∼19 when the two T50E mutants are compared. Therefore, the Lu cofactor seems to be less 13101



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acid, T50, to a tryptophan renders both cofactors from light active to light inactive. On the other hand, the photoreduction kinetics of T50E is in the same range as that of the wild type, either with a ribolumazine or the Rf cofactor. In addition, the T50E mutant harboring a Rf cofactor shows a time-resolved signal both with optical and with trEPR detection that can be assigned to a flavin triplet state. The different behavior of the two mutants can be rationalized by a significant modulation of the excited states by the aromatic tryptophan residue close to the flavin cofactor: As already published for a number of other flavoproteins, aromatic amino acids such as tyrosines or tryptophans are able to alter the lifetimes and the energy of any excited state (see, e.g., ref 78) and, thus, favor the radiationless relaxation back to the ground state. We assume that this effect also occurs in the T50W mutant. The two remaining mutants S48C and N101W have in common that both amino acids interact with the ribityl side chain but not directly with the heteroaromatic ring systems. Specifically, S48 is located beneath the pyrazine ring and forms a hydrogen bond to the 2′-hydroxy group. N101, on the other hand, is located close to the 3′-hydroxy group of the ribityl side chain and forms a hydrogen bond with it. Quite unexpectedly, the photoreduction kinetics of N101W is, independent of the cofactor, the slowest of all investigated samples, even though the tryptophan residue is located more distant to the redoxactive part of the cofactors than in the T50W mutant. We believe that the tryptophan alters the cofactor binding site to a significant extent and consequently determines the photophysics to a redox-inactive state. It is also worth mentioning that both the N101W and the S48C mutants harboring the Rf cofactor accumulate a radical state upon light illumination. To summarize, efficient electron transfer for the reduction of both the Rf and the ribolumazine cofactor is possible; however, no indications of any involvement of amino acids are observed. Therefore, a direct electron transfer from the solvent to the cofactor seems most likely. EPR Spectroscopy. Another focus of this contribution was to provide a first comprehensive EPR characterization of the Lurad redox state in LumP WT. For an overall analysis of the Lurad redox state, W-band EPR spectra were recorded both in protonated and in deuterated buffer solutions. Although the anisotropy of the g-tensor was still not fully resolved, good spectral simulations of the experimental data were obtained. Surprisingly, the anisotropy of the Lu-LumP g-tensor is similar to that of other flavoproteins: Principal components in the range of gx = 2.004, gy = 2.0035, and gz = 2.002 have been published for a variety of neutral flavoprotein radicals (see, e.g., refs 58 and 62); the principal components for ribolumazine in LumP obtained in this study are gx = 2.0043, gy = 2.0034, and gz = 2.0020. Additionally, two (axially symmetric) hfc tensors from the nitrogens N(5) and N(8) have to be included for a good spectral simulation. The principal values of 42.2 MHz, 0 MHz, 0 MHz and 36.6 MHz, 0 MHz, 0 MHz demonstrate the strong axiality of the two nitrogen tensors. The main component of the latter tensor is significantly larger than the respective ones in flavin radicals (which are in the range of 27− 30 MHz for neutral flavin radicals)65 and reflects the altered spin density distribution of Lu as compared to Rf derivatives. Additional information about the spin-density distribution in LumP containing the ribolumazine cofactor is expected to result from analysis of ENDOR experiments. Here, Davies-type ENDOR has been performed to unravel the proton hfcs and compare the results with those obtained from LumP samples

suited for redox-state changes as compared to the Rf cofactor. These findings are further supported by time-resolved optical data: Whereas only fast-decaying fluorescence can be detected in LumP samples harboring the ribolumazine cofactor, significant amounts of long-lived triplet states and even a small amount of excited flavin radical are detected (see Figure 4). In principle, these findings could be explained by the (i) similar but nevertheless different chemical nature of the two cofactors, (ii) markedly different redox potentials of the two cofactors, and/or (iii) different protein−cofactor interactions modulating the photophysical properties of the respective cofactors. The redox potential of Rf semiquinone formation has been determined as −313 mV versus NHE (pH = 7.0) in aqueous solution.76 In comparison, the redox potential for the reduction of 8-methyllumazine in aqueous solution was published as −424 mV versus NHE (−621 mV versus Ag/AgCl).77 Both values are in the same range but show a difference of ∼100 mV. A direct comparison of the two values and its implication for LumP photoreduction kinetics, however, is not easily possible as (i) both values have been obtained for the free cofactors in aqueous solution and (ii) no experimental values of the cofactor used in our experiments, ribolumazine, have been published yet. From a structural perspective, both cofactors are located at the surface of LumP in the same binding pocket, forming a similar hydrogen-bond network (see Figure 1B/C and PDBIDs 3A3G, 3A3B, and 3DDY for details).16,35 Whereas the pyrimidine ring is tightly bound by hydrogen bonds from surrounding amino acids (especially by amino acids T50, D62, and D64), the two methyl groups 6α and 7α in ribolumazine (7α and 8α in Rf) are protruded from the surface into the bulk solvent. Therefore, no defined interactions between this side of the two cofactors and any amino acids are presumed, and a direct contact between solvent molecules and the cofactor is plausible. We thus can conclude that the observed changes in the photoreduction kinetics of LumP are most likely not due to differences in the redox potentials and/or structural reasons but in fact are due to their different chemical nature. Additionally, we presume that any external electron donor is also able to interact directly with the cofactor, and thus, photoreduction occurs directly between the electron-donor molecule and the cofactor. With this assumption, we will also attempt to elucidate the behavior of the four investigated mutant samples. These can be divided into several groups: The first contains the two mutants of threonine 50, namely, T50W and T50E. Threonine-50 is one of the amino acids responsible for efficient cofactor interaction. Specifically, T50 forms two hydrogen bonds to the oxidized cofactors, one from the 3-hydroxyl group to N(5) and the other from the 2-amino group to O(4) (Figure 1). Therefore, exchange of this threonine either to the bulky, charged glutamic acid or to the bulky, aromatic tryptophan is supposed to change the hydrogen-bond situation and, thus, the photophysics significantly. Interestingly, the effect of the mutations points to completely different directions: The reduction speed is dramatically reduced for the T50W mutant, independent of the cofactor used, and no radical states of the cofactors are accumulated (Figure 3). Additionally, the T50W mutant shows no signal in any time-resolved spectroscopies, neither at room temperature using optical detection nor at low temperature using trEPR (Figure 4 and Figure S1, Supporting Information). These findings demonstrate that the exchange of one amino 13102



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present study and will be the subject of a subsequent publication. Photoreduction has been repeated with four mutants, and their kinetics are altered significantly. Special emphasis must be given to the tryptophan mutant T50W; here, no time-resolved signals and a drastically reduced photoreduction speed are observed. Tryptophan residues in the vicinity of the chromophore are obviously able to shift the protein from light active to light inactive. Moreover, the Lu-LumP radical state was investigated by Wband EPR and X-band ENDOR spectroscopies. Both the principal values of the g tensor and an almost complete mapping of the proton hfcs could be achieved. Remarkably, the g-tensor’s principal components are similar to the respective Rfcontaining protein; however, the proton hfcs show noticeable differences, which are not unexpected due to the different chemical nature of the cofactors. In summary, ribolumazine-containing LumP can be easily converted from the oxidized to its other redox states via photoreduction, and it forms a metastable neutral radical under anaerobic conditions. On a fast time scale, however, the ribolumazine-containing protein seems to be optimized for high fluorescence yield, whereas the Rf-containing proteins show a high yield of triplet state. Hence, the function of ribolumazine as redox-active cofactor with similar but not identical properties as compared to Rf seems plausible; however, the existence of other proteins harboring ribolumazine waits to be proven.

containing the Rf cofactor. Key for the assignment of proton hfcs is the comparison of samples measured in protonated and deuterated buffers and the electron-spin density maps obtained from DFT calculations. Three types of protons, namely, the ones attached to C(7α) and the two protons attached to N(3) and N(5), are supposed to be acidic and, thus, susceptible to deuterium exchange. With this knowledge at hand, the hfcs of all protons attached to the spin system could be unambiguously assigned (Table 2). In this context, the Lu radical could be clearly identified as a neutral, N(5)-protonated radical. In Rf-LumP, a number of larger hfcs could be assigned, namely, those from the protons H(1′), H(5), and H(8α); all smaller hfcs are unfortunately obscured by overlapping resonances in the X-band ENDOR spectra and consequently could not be assigned by this type of spectroscopy. Qualitatively, the hfcs from Rf-LumP are comparable to those obtained from other flavoproteins.79 Upon closer inspection, the Aiso values of the protons H(1′) and H(8α) are more related to those obtained in flavodoxins than in photolyases and other flavoproteins.79 This finding can be rationalized in terms of a different flavin-binding situation. Whereas the flavin cofactor is deeply buried in, e.g., photolyases, it is located near the surface of the protein in flavodoxins, with the xylene ring protruding into the solvent. Therefore, the strength of the hfcs of these protons reflects an extremely polar aqueous environment. On the other hand, the hfcs differ significantly when comparing them with those obtained in Lu-LumP. Qualitatively, the principal hyperfine values of the protons H(1′) and H(7α) are considerably larger in Lu-LumP, which can be explained by a significant shift in electron spin distribution in Lu-containing as compared to Rf-containing LumP samples. On the other hand, the Aiso value for H(5) is decreased by ∼10 MHz, which is caused by (i) the change in sign for A1 and (ii) by a significantly smaller anisotropy of the H(5) hfc. These findings point toward a significantly altered singly occupied molecular orbital with shifted spin densities that reflect the different chemical nature of the two cofactors (see Figure S2, Supporting Information, for a representation of the singly occupied molecular orbitals of the ribolumazine model).

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Detailed analysis of the transient absorption data; fitting of the transient EPR data; and DFT calculations are available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.W. and E.S. thank Robert Bittl (Free University Berlin) for use of some EPR equipment, Deborah Meyer for help with the triplet simulation, and the DFG (RTG1976, project P13) for funding. M.F. and S.W. thank the DFG (WE 2376/4-1 and FI 824/6-1) for funding.

CONCLUSIONS In this article, we investigated the lumazine protein including its biologically active cofactor, ribolumazine, in different redox states and compared the results with samples containing a Rf cofactor. Using anaerobic photoreduction, we were able to record different kinetics from both cofactors in similar protein environments and characterize them in terms of protein− cofactor interactions using a variety of mutants. It could be demonstrated that LumP is able to stabilize a neutral ribolumazine radical with a ∼35% yield; however, solely fluorescence but no indications of any intermediate radical or a triplet state could be obtained using time-resolved spectroscopy. This is in contrast to LumP samples containing the Rf cofactor, where a neutral radical could be obtained, and a high yield of excited triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. Therefore, it seems possible that Lu-LumP limits electron transfer and inhibits intersystem crossing, thus rendering the protein optimized for fast and efficient singlet-state decay. It would be highly interesting to see if this photophysical behavior can be modified in the presence of its natural interaction partner, the luciferase protein. This is, however, beyond the scope of the

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REFERENCES

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One Protein, Two Chromophores: Comparative Spectroscopic

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